An important challenge in developmental biology is to understand the molecular mechanisms that establish body form during embryogenesis. In Drosophila, the process of segmentation subdivides the embryo into a series of repeated units (parasegments) along the anterior-posterior axis. This process is controlled by at least 30 genes, which comprise a transcriptional hierarchy that converts periodic signals generated by maternal gradients and gap gene expression domains into patterns of increasingly refined stripes. Drosophila is an excellent system for studying interactions between proteins within a regulatory network because changes in expression patterns can be directly visualized in the embryo. This proposal will focus on the trans- and cis- acting components involved in the transcriptional regulation of the seven striped patterns of the pair-rule genes. In the first part, genetic manipulations and a targeted mis-expression strategy will be employed to change the levels and positions of the gap gene expression domains. The objective of these experiments is to test whether these factors function as gradient morphogens to set up pair rule-patterns. The second part will focus on the cis-regulatory components that control the expression of the pair-rule patterns. The second part will focus on the cis-regulatory components that control the expression of the pair-rule gene even-skipped (eve). Previous studies have identified two approximately 500 bp enhancers that independently regulated stripe 2 and stripe 3 of eve's seven stripe pattern. These enhancers act as genetic switches that control transcriptional activation and repression by integrating the positional information provided by the gap genes. Most of these experiments will focus on the detailed mechanisms mediated by the stripe 2 enhancer. First, in vitro binding assays will be performed to identify interactions between regulatory proteins at the level of the enhancer that may control the transcriptional state of the enhancer. Second, a combination of in vitro and in vivo approaches will be used to identify binding sties for unidentified factors that may important for stripe 2 enhancer function. Third, the role of binding site number, affinity, and spacing of stripe 2 regulation will be tested in vivo by P-element-mediated transformation. Finally, P-element transformation assays will be used to determine how spacer sequences between the stripe 2 and stripe 3 enhancers insure the autonomous activity of each enhancer. These studies should contribute significantly to our understanding of how gradients control pattern formation, and how eukaryotic enhancers in general activate and repress transcription.